Download M-cells: origin, morphology and role in mucosal immunity and

MINIREVIEW
M-cells: origin, morphology and role in mucosal immunity and
microbial pathogenesis
Sinead C. Corr1, Cormac C.G.M. Gahan1,2 & Colin Hill1
1
Department of Microbiology, Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland; and 2School of Pharmacy, University College
Cork, Cork, Ireland
Correspondence: Sinead C. Corr,
Department of Microbiology, Alimentary
Pharmabiotic Centre, University College Cork,
Cork, Ireland. Tel.: 1353 21 901712;
fax: 1353 21 4903101; e-mail:
[email protected]
Received 12 April 2007; revised 26 October
2007; accepted 30 October 2007.
First published online 11 December 2007.
DOI:10.1111/j.1574-695X.2007.00359.x
Editor: Willem van Leeuwen
Abstract
M-cells are specialized cells found in the follicle-associated epithelium of intestinal
Peyer’s patches of gut-associated lymphoid tissue and in isolated lymphoid
follicles, appendix and in mucosal-associated lymphoid tissue sites outside the
gastrointestinal tract. In the gastrointestinal tract, M-cells play an important role in
transport of antigen from the lumen of the small intestine to mucosal lymphoid
tissues, where processing and initiation of immune responses occur. Thus, M-cells
act as gateways to the mucosal immune system and this function has been
exploited by many invading pathogens. Understanding the mechanism by which
M-cells sample antigen will inform the design of oral vaccines with improved
efficacy in priming mucosal and systemic immune responses. In this review, the
origin and morphology of M-cells, and their role in mucosal immunity and
pathogenesis of infections are discussed.
Keywords
M-cells; translocation; bacteria; pathogens.
Introduction
The mucosal-associated lymphoid tissue (MALT), consisting of immunoreactive cells and organized lymphoid tissues,
is found in close contact with all mucosa throughout the
body. In the intestine, it is termed gut-associated lymphoid
tissue (GALT), which consists of both isolated and aggregated lymphoid follicles (Neutra et al., 2001). These are
the sites where antigen recognition and mucosal immune
responses are initiated. Aggregated lymphoid follicles are
found in Peyer’s patches (PP) of the small intestine, appendix vermiformis, and caecum, colon and rectum patches.
GALT is one of the largest lymphoid organs in the body,
containing up to 70% of the body’s immunocytes. Typical
GALT structures can be seen in PP, aggregated lymphoid
follicles in the small intestinal mucosa. PP are named after
the 17th-century Swiss anatomist Hans Conrad Peyer and
are located along the antimesenteric side of the small
intestine. Morphologically, PP are separated into three main
domains: the follicular area, the parafollicular area and the
follicle-associated epithelium (Neutra et al., 2001). The
follicular and parafollicular areas consist of the PP lymphoid
follicle, which has a germinal centre containing proliferating
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B-lymphocytes, follicular dendritic cells (FDC) and macrophages. The follicle is surrounded by the corona, containing
small lymphocytes expressing IgM and IgD; a dome lies above
the follicle and contains B- and T-lymphocytes, dendritic cells
(DCs) and macrophages. The follicle-associated epithelium
(FAE) is a one-cell-thick layer composed of enterocytes and
specialized epithelial cells termed M-cells (Owen & Jones,
1974). The FAE overlies the PP and forms the interface
between the intestinal lymphoid system and the intestinal
luminal environment. The function of PP was unknown
until 1922, when Kenzaburo Kumagai identified uptake of
Mycobacterium tuberculosis at the dome epithelium. However,
he also observed uptake of heat-killed bacteria, sheep red
blood cells and India ink by PP and hence concluded that this
uptake was a nonspecific process. With the development of
techniques for ultrastructural analysis in 1972, M-cells capable of taking up antigen were identified and the role of PP in
the immune system became clear (Owen & Jones, 1974).
M-cells
M-cells are specialized epithelial cells found in the FAE of
PP, and in isolated lymphoid follicles, appendix, and in
FEMS Immunol Med Microbiol 52 (2008) 2–12
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M-cells
MALT sites outside the gastrointestinal tract. M-cells differ
morphologically and enzymatically from adjacent enterocytes. In 1965, M-cells were first identified in rabbit appendix by J.F. Schmedtje (Owen & Jones, 1974). They were
initially called lymphoepithelial cells but were later renamed
M-cells, when Owen & Jones (1974) used electron microscopy
to study PP of human small intestine and found the presence
of ‘microfolds’ on the apical surface of these epithelial cells.
M-cells act as gatekeepers to the mucosal immune system,
continuously sampling the lumen of the small intestine and
transporting antigen to the underlying mucosal lymphoid
tissue for processing and initiation of immune responses
(Kraehenbuhl & Neutra, 1992; Neutra et al., 1996a, b). This
M-cell sampling process has been exploited as a means of
translocating the epithelium by pathogens including Salmonella typhimurium (Jensen et al., 1998).
DiRita, 2006a). The presence of M-like cells in untreated
Caco-2 monolayers was confirmed by the observation of
cells lacking an organized brushborder and the presence of
the M-cell marker, sialyl-Lewis A antigen (Blanco & DiRita,
2006a).
Combining histochemical and ultrastructural techniques,
Gebert et al. (1999) analysed PP dome epithelium development and found M-cell precursor cells in specialized domeassociated crypts. These crypts differ from ordinary crypts
in size, shape, cellular composition and location. Thus, this
study suggests that M-cell differentiation is restricted to
specialized crypts and is not induced by lymphocytes.
Furthermore, a study by Gebert et al. (1999) detected M-cell
precursors in the early proliferative zone of the crypts using
M-cell-specific monoclonal antibodies. This observation
again supports the theory that M-cells arise from a distinct
cell lineage.
Origin and development of M-cells
The origin of M-cells within the FAE remains unclear and
is the subject of much debate. It is known that intestinal
epithelial cells in the FAE originate from stem cells in crypts
located between a villus and a PP dome. Each crypt
harbours a ring of stem cells that generate distinct cell types
and there are two distinct axes of migration and differentiation (Heath, 1996). Cells located on the villous side of the
crypt differentiate into absorptive enterocytes, goblet cells
and endocrine cells as they migrate upwards in columns
along the villous (Sierro et al., 2000). Cells enter programmed cell death as they reach the tip of the villous and
are shed into the intestinal lumen (Sierro et al., 2000). Cells
on the FAE side of the crypt move into the dome and
differentiate into absorptive enterocytes and M-cells (Garvrieli et al., 1992).
While it is accepted that enterocytes and M-cells have
a common precursor, it is not known whether crypt cells
commit early to FAE and M-cell phenotypes, or whether
factors act later to induce further differentiation of enterocytes into M-cells (Nicoletti, 2000). Certainly, an established in vitro coculture system demonstrates that human
intestinal enterocytes are converted to M-like cells by
murine Peyer’s patch lymphocytes (PPL) (Kerneis et al.,
1997). In this in vitro model, PPL migrate into spaces
between epithelial cells, forming intraepithelial pockets in
enterocytes with disordered apical microvilli, characteristic
features of an M-cell (Kerneis et al., 1997). However,
whether this reflects the mechanisms governing in vivo
differentiation is unclear as the in vitro system utilizes
adenocarcinoma cells (Caco-2) that do not behave like
normal enterocytes. Furthermore, a recent study found that
prior treatment of Caco-2 epithelial monolayers with a
human lymphocyte cell line is not absolutely required for
differentiation to the M-like cell phenotype (Blanco &
FEMS Immunol Med Microbiol 52 (2008) 2–12
M-cell morphology
M-cells are distinguished from intestinal enterocytes by
distinctive morphological features. At the apical surface,
M-cells display a poorly organized brush border with short
irregular microvilli, in contrast to the highly organized
brush border of enterocytes, with uniform densely packed
microvilli (Figs 1 and 2) (Kerneis et al., 1997). Immunostaining of the microvillar proteins, actin and villin, has been
used to identify M-cells that are detected by the absence of
staining due to their short, irregular microvilli (Kanaya
et al., 2007). The usually thick glycocalyx associated with
absorptive cells is absent in M-cells and is replaced by a thin
glycocalyx, which is thought to aid greater access to antigens
in the gut lumen. M-cells lack certain enterocyte apical
surface glycoproteins. These include alkaline phosphatase
and sucrase-isomaltase activity, which are typical to the
brushborder of enterocytes, and both have been used as
negative markers for M-cells (Gebert et al., 1996). A lack of
Fig. 1. Electronmicrograph of an M-cell from a Caco-2 coculture experiment displaying short irregular microvilli (Corr et al., 2006).
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4
S.C. Corr et al.
(a)
Villous
Villi
Follicleassociated
epithelium
Lymphoid
follicle
Crypts
Draining
lymphatic
vessels
(b)
M cell
apical
basolateral
T lymphocyte
B lymphocyte
Dendritic cells
&
Macrophages
(Giannasca et al., 1999). These lectin-binding sites were also
observed on M-cell plasma membranes, including the
basolateral membrane, pocket domain and on intracellular
vesicles (Neutra et al., 1999). Furthermore, glycosylation
patterns vary between M-cells within a single FAE, at
different intestinal locations and between species (Gebert &
Hach, 1993). Rabbit caecal M-cells selectively bind UEA-1
but UEA-1 is not bound by rabbit PP and M-cells in mouse
caecal patches (Jepson et al., 1996). Also, human colonic
M-cells express intercellular adhesion molecule 1 (ICAM-1)
but it is not expressed by PP M-cells (Jepson et al., 1996).
Research is ongoing to determine the cellular markers of
human M-cells. Significantly, recent work has utilized
microarray analysis of Caco-2 cells differentiated to M-like
cells and determined 180 differentially regulated genes. Of
these potential targets, the molecule Galectin 9 was shown to
be expressed on the apical surface of M-like cells and FAE
and may provide a molecular marker of human M-cell
differentiation (Pielage et al., 2007). Monoclonal antibodies
that are specific against single carbohydrate epitopes have
also been used to identify M-cells in humans (Neutra et al.,
1999). Using this approach, M-cells have been shown to
express sialyl Lewis antigen on their apical and subcellular
membranes (Giannasca et al., 1999).
M-cell function
Fig. 2. Overview of M-cell location within the PP FAE.
alkaline phosphatase has been used to signify M-cells in
mice, rabbits, rats, dogs and humans (Gebert et al., 1996). At
their basolateral surface, M-cells possess a unique intraepithelial invagination or ‘pocket’ (Neutra et al., 1996a). The
M-cell pocket contains B- and T-lymphocytes, macrophages
and DCs. It provides a docking site for lymphocytes and
other antigen-presenting cells, reducing the distance from
the apical to the basolateral surface that transcytotic vesicles
travel (Trier, 1991). The basolateral surface of epithelial cells
contains two major subdomains: the lateral subdomain is
involved in cell–cell adhesion and contains Na1–K1–ATPase, and the basal subdomain interacts with the extracellular matrix and the lamina propria (Trier, 1991).
While little is known about the glycoproteins or adhesion
molecules expressed on the apical surface of M-cells, M-cells
have been shown to display distinct glycosylation patterns as
compared with their enterocyte neighbours (Gebert & Hach,
1993). Lectins have been used to characterize this glycosylation pattern. Ulex europaeus 1 (UEA-1) and Psophocarpus
tetragonolobus (WBA) are two lectins that have been used
to stain murine M-cells (Giannasca et al., 1994). UEA-1 is
specific for carbohydrate structures that contain a-1-fucose
residues, and specifically stains the apical surfaces of M-cells
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The reduced brush border and lack of enzymatic activity of
the M-cell apical surface suggests that they are unlikely to
play a role in digestion or absorption. The exposed nature of
the M-cell apical surface instead indicates that the primary
function of M-cells is trans-epithelial transport. M-cells
transport substances from the lumen of the intestine, across
the epithelial barrier and to underlying immune cells where
processing and initiation of immune responses occur.
M-cells have been shown to transport particulates including
latex beads, carbon particles and liposomes and macromolecules including ferritin, horseradish peroxidase, cholera
toxin-binding subunit, lectins and antivirus antibodies
(Gebert et al., 1996). M-cells have also been shown to
transport microorganisms including Vibrio cholerae and
S. typhimurium in vitro (Kerneis et al., 1997; Jensen et al.,
1998). This transport process occurs via trans-cellular
endocytosis and has been shown to be temperature-dependent, transport being inhibited at 25 1C or lower (DesRieux
et al., 2005).
The trans-epithelial transport process occurs in three
stages. Firstly, endocytosis of the substance occurs at the
apical membrane, followed by transport of the substance via
an endocytic vesicle to the endosomal compartment, and
finally exocytosis at the basolateral membrane (DesRieux
et al., 2005). M-cell-mediated-translocation is very efficient
and is a rapid process. As the M-cell cytoplasm above
FEMS Immunol Med Microbiol 52 (2008) 2–12
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M-cells
the pocket is a thin apical rim, a complete endocytosis–
exocytosis sequence can take a minimum of ten minutes
(Kraehenbuhl & Neutra, 1992). The mechanisms by which
M-cells take up microorganisms and molecules vary according to the nature of the material such as size, local surface
pH, surface charge, hydrophobicity, concentration and the
presence or absence of an M-cell-specific receptor (DesRieux et al., 2005). For example, large particles and bacteria
can induce phagocytosis associated with apical membrane
ruffling and rearrangement of the actin cytoskeleton (Liang
et al., 2001). Viruses and other adherent particles are taken
up by endocytosis via clathrin-coated vesicles (Owen, 1977).
Nonadherent material is taken up by fluid-phase endocytosis and this has been shown in the case of soluble tracers
such as ferritin (Neutra et al., 1987). A study by Neutra et al.
(1987) found that a membrane-bound tracer, wheat germ
agglutinin (WGA) lectin, is transported by M-cells c. 50
times more efficiently than the soluble tracer, bovine serum
albumin (BSA).
During transcytosis, antigens do not undergo major
ultrastructural alteration and are released intact into the
pocket. While M-cells have a 16-fold decrease in lysosome
volume and reduced lysozyme (acid phosphatase) activity
compared with normal enterocytes, there is some evidence
that M-cells may possess some enzymatic activity. Acid
phosphatase-enriched prelysosomes, lysosome vesicles and
major-histocompatability complex (MHC) class II determinants in the basolateral domain were detected in rat M-cells,
suggesting a role in antigen processing and presentation
(Allan et al., 1993). The aspartic proteinase Cathepsin E,
normally found in the lysosomal compartment of antigenpresenting cells, has been localized in human and rat Mcells, again suggesting a role in processing and presentation
(Finzi et al., 1993). Despite these observations, the extent of
degradation and participation of M-cells in antigen processing and presentation remains unclear.
M-cells have other roles apart from antigen transport. It
has been suggested that M-cells may aid immune response
induction to the antigen they are transporting by releasing a
costimulatory signal for T- and B-cell proliferation (Pappo
& Mahlman, 1993). M-cells isolated from rabbit FAE were
shown to secrete IL-1 following lipopolysaccharide stimulation in vitro and these culture supernatants were shown to
induce T-cell proliferation (Pappo & Mahlman, 1993). This
suggests that transcytosis of bacteria results in bacteriallipopolysaccharide-induced IL-1 release, aiding proliferation of lymphocytes for induction of a mucosal immune
response.
Entry sites for pathogens
M-cells are the main sites for continuous sampling and
transport of antigens from the intestinal lumen to mucosal
FEMS Immunol Med Microbiol 52 (2008) 2–12
lymphoid tissues. Despite the production of antimicrobial
peptides at the M-cell gateway (Goitsuka et al., 2007), this
region of the FAE can be considered to be a potential
‘Achilles heel’ in the mucosal barrier and M-cells are
exploited by many pathogens as a route of entry to underlying host tissues (Sansonetti & Phalipon, 1999). These
include Poliovirus, S. typhimurium, Yersinia enterocolitica
and V. cholera (Kerneis et al., 1997; Jensen et al., 1998;
Ouzilou et al., 2002; Hamzaoui et al., 2004). As M-cells are
capable of transporting inert particles, it has been suggested
that pathogens may interact with M-cells via nonspecific
passive mechanisms. However, several studies indicate that
specific mechanisms of interaction mediate transport of
microorganisms by M-cells (Tyrer et al., 2007). Understanding the mechanisms by which some microorganisms selectively use M-cells to cross the intestinal epithelium may aid
development of disease control strategies. It may also allow
exploitation of these invasion strategies for mucosal drug
and vaccine delivery (Brayden et al., 2005).
Salmonella
Salmonella spp. are probably the most-studied pathogens
that translocate M-cells. Murine ligated loop models have
shown that M-cells are the major route of entry for
Salmonella (Jones et al., 1994). Salmonella typhimurium
exhibits selective targeting of M-cells overlying PP and can
induce uptake, which is associated with extensive ruffling
and FAE damage (Sansonetti & Phalipon, 1999). Adherence
and invasion of M-cells induces apical membrane ruffling
and actin polymerization, leading to bacterial engulfment,
M-cell cytotoxicity and sloughing of the FAE (Jones et al.,
1994). This FAE damage could potentially allow unrestricted
bacterial invasion and may explain the occurrence of intestinal ulcers and perforations in typhoid patients (Jones
et al., 1994). Rearrangements of FAE were exclusively
observed at sites of M-cells in mice 30 min postinfection
with S. typhimurium, with the integrity of enterocytes
remaining intact. With increased length of exposure, increased M-cell membrane alterations were observed, with
most M-cells displaying membrane protrusions and disruptions after 90 min. These disruptions result in pores in the
epithelium that allow bacterial spread to organs before an
immune response is established (Jones et al., 1994). The
invasion machinery, encoded by genes located on the
Salmonella pathogenicity island 1 (SPI1), is crucial for
invasion of epithelial cells and also plays a role in M-cell
invasion (Clark et al., 1998b). However, while mutants in
SPI1 still invade M-cells they, are not cytotoxic and do not
result in the destruction of the FAE (Jones et al., 1994).
Salmonella typhimurium expresses a number of fimbrial
operons, including fim, lpf and pef. These mediate binding
to different murine epithelial cells and the lpf operon
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appears to be responsible for specific adherence to murine
M-cells (Baumler et al., 1996). An S. typhimurium mutant in
lpfC exhibited reduced colonization of PP and reduced
destruction of M-cells, suggesting that S. typhimurium target
M-cells via these fimbriae (Baumler et al., 1996). Following
transport across the FAE, Salmonella are phagocytosed by
macrophages and DCs where they can survive within the
phagocytic vacuole due to genes encoded by a second
pathogenicity island (SPI2) (Baumler et al., 1996).
Yersinia
Microscopic studies in mice have shown that Yersinia
enterocolitica and Yersinia pseudotuberculosis adhere to both
enterocytes and M-cells, but demonstrate a preference for
M-cells (Autenrieth & Firsching, 1996; Marra & Isberg,
1997; Sansonetti & Phalipon, 1999). Infection with
Y. enterocolitica is accompanied by major damage to PP
(Autenrieth & Firsching, 1996). It has been demonstrated
that targeting of Yersinia to M-cells is mediated by invasin,
an outer-membrane protein that binds to integrins of the b1
family expressed apically on M-cells (Autenrieth & Firsching, 1996; Marra & Isberg, 1997; Clark et al., 1998a). Despite
a recent study that showed that an invasin-deficient strain
can still adhere to M-cells in vitro, Yersinia mutants lacking
invasin protein display reduced colonization and translocation of PPs in vivo (Marra & Isberg, 1997; Hamzaoui et al.,
2004). Following M-cell uptake, bacteria must survive
phagocytosis by macrophages. Yersinia have developed a
mechanism of antiphagocytosis that allows them to remain
extracellular (Forsberg et al., 1994). This strategy is shared
by the enteropathogenic Y. enterocolitica and Y. pseudotuberculosis, and also the plague organism Yersinia pestis. Following adherence to the eukaryotic cell surface, the bacterium
injects a set of Yop proteins (Yop-E, -H, -T, -O, -P and -M)
into the cytoplasm via a Type III secretion system (Grosdent
et al., 2002). These Yop proteins induce breakdown of the
F-actin network and Yop-O, also called YpkA, interrupts
signalling pathways of phagocytosis (Forsberg et al., 1994).
S.C. Corr et al.
membrane ruffling; however, S. flexneri are not cytotoxic to
M-cells (Perdomo et al., 1994; Jensen et al., 1998, Sansonetti
& Phalipon, 1999). Following passage across the FAE,
bacteria reinvade epithelial cells basolaterally and invasion
is followed by an inflammatory process that disrupts the
epithelium and increases permeability, facilitating further
passage across the FAE (Perdomo et al., 1994). Bacteria are
then engulfed by macrophages and DCs. Two to four hours
following infection, bacteria induce apoptosis by expressing
four ipa genes encoding secretory proteins and an Mxi/Spa
secretory apparatus. Bacteria release an IpaB invasin that
binds to IL-1b-converting enzyme, causing cleavage of
target proteins and cell death. Upon apoptosis, bacteria are
released and spread from cell to cell, inducing the production of proinflammatory cytokines, IL-8 and TNF-a by
enterocytes (Sansonetti & Phalipon, 1999; Phalipon &
Sansonetti, 2007).
Vibrio cholerae
Binding of V. cholerae to M-cells occurs via a tight attachment domain that induces actin filaments to form structures
that engulf the bacteria. Heat-killed bacteria do not attach
to M-cells, indicating that the interaction requires specific
bacterial adhesions (Blanco & DiRita, 2006a). A recent study
found that V. cholerae is transcytosed by M-cells via interaction of cholera toxin and ganglioside receptor, GM1 (Blanco
& DiRita, 2006a, b). In this study, heat-killed bacteria were
not transcytosed as heat-killing may destroy or remove
cholera toxin required for binding to GM1. Following
transport across M-cells, V. cholerae infect lymphoblasts
and macrophages, but this is accompanied by subsequent
killing of the bacteria in the follicle dome (Davis & Owen,
1997). Uptake induces a host sIgA response to cholera toxin
and the cholera lipopolysaccharide (Pierce et al., 1987).
However, pathogen-bound sIgA may then further enhance
M-cell-mediated uptake of V. cholerae through targeting of
M-cell sIgA receptors (Blanco & DiRita, 2006b).
Escherichia coli
Shigella flexneri
Invasion of the intestinal epithelium by Shigella spp. causes
the acute infection termed recto-colitis, better known as
shigellosis. Inflammatory lesions occur on the upper rectum
and distal colon. Upon further histopathological study,
these ulcers were found to be located at sites of lymphoid
follicles, suggesting that the FAE is a major route of entry
(Sansonetti et al., 1996). In rabbit ligated loop assays,
S. flexneri enter M-cells after a short infection period
(2–8 h), causing an increase in M-cell size due to accumulation of mononuclear cells in the pocket and thus acts as an
increased area for transport (Perdomo et al., 1994). This
process is accompanied by extensive cellular damage and
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Most strains of Escherichia coli do not adhere to M-cells,
with the exception of some pathogenic strains. These
include E. coli 0:124, enteroaggregative and diffuse adhering
E. coli, enterotoxigenic E. coli and enteropathogenic E. coli
rabbit diarrhoeagenic E. coli (RDEC)-1 (Cantey & Inman,
1981). The RDEC-1 strain of E. coli is an enteroadherent
bacterium that adheres to the lymphoid follicle epithelium
of ileal PP in postweanling rabbits (Inman & Cantey, 1983).
In an ultrastructural study, E. coli RDEC-1 were seen to
adhere specifically to M-cells of the lymphoid follicle
epithelium; however, this did not result in their internalization (Cantey & Inman, 1981). Adherence is accompanied by
blebbing and notching of the M-cell microvilli leading to the
FEMS Immunol Med Microbiol 52 (2008) 2–12
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M-cells
eventual formation of vesicles. Adherence to absorptive
epithelial cells causes pedestal formation by the plasmalemma that cup the bacteria (Cantey & Inman, 1981; Marchetti
et al., 2004). A recent study found that nonmotile E. coli
RDEC-1 do not efficiently adhere to appendix M-cells of the
rabbit (Marchetti et al., 2004). The enterohaemorrhagic E.
coli strain O157:H7 selectively adheres to human FAE PP but
it is not known whether it specifically adheres to M-cells.
However, the protein intimin (g), essential for its colonization, binds b1-integrins, and these are expressed on the
M-cell apical surface (McKee et al., 1995; Hamzaoui et al.,
2004). A recent study observed similar rates of translocation
of enteropathogenic E. coli O127:H7 across an in vitro Mcell model compared with normal enterocytes (MartinezArgudo et al., 2007). Furthermore, in this study, it was
observed that translocation rates were significantly increased
in the absence of a functioning TypeIII secretion system
(Martinez-Argudo et al., 2007).
Listeria
Considerable research work has centred on the precise route
by which the food-borne pathogen Listeria monocytogenes
gains access to Intraepithelial lymphoid cells and mucosal
lymphoid tissues. It has been well documented that
L. monocytogenes can invade nonphagocytic cells (using
bacterial internalin proteins) and that this process is critical
for bacterial translocation of the intestinal epithelium
(Vázquez-Boland et al., 2001; Hamon et al., 2006). Pentecost
et al. (2006) have recently demonstrated that the pathogen
binds to E-cadherin exposed at the villous tips during
extrusion of epithelial cells in this region. Previous work
has demonstrated that this interaction is host specific and
that human, but not murine and rat cells are efficiently
targeted by L. monocytogenes (Lecuit et al., 1999).
While it is clear that the pathogen invades host cells
through a specific interaction between internalins and target
receptors, some speculation surrounds whether the pathogen also has the potential to invade via M-cells for rapid
translocation. This interaction would effectively prime the
host immune response but could also be utilized by the
pathogen as a means for accessing the underlying mucosa.
Indeed, some evidence for M-cell sampling of L. monocytogenes has emerged from studies in mice and rats (Pron et al.,
1998; Daniels et al., 2000). In vivo analysis of orogastric
L. monocytogenes infections using animal models has
demonstrated preferential replication within the PP with
extremely rapid translocation of the bacterium (within
15 min) to internal organs (Pron et al., 1998). Similarly,
a more recent study using the murine model of infection
(Daniels et al., 2000) found very rapid translocation of
Listeria from the lumen of the gastrointestinal tract to
internal organs. Other studies also demonstrated rapid
FEMS Immunol Med Microbiol 52 (2008) 2–12
localization of L. monocytogenes within the PP of mice
(Marco et al., 1997; Corr et al., 2006). It is evident that in
the absence of a specific interaction between L. monocytogenes and murine host cells, the pathogen may be sampled
within PP, providing for rapid translocation.
The in vitro model of M-cell development was recently
utilized for analysis of L. monocytogenes translocation. This
work demonstrates that the pathogen migrates through
differentiated M-cells at rates similar to translocation of
V. cholerae and more efficiently than control Lactobacillus
salivarius cells. Furthermore, this interaction is independent
of internalin expression or expression of the major virulence
factor Listeriolysin (Corr et al., 2006). Using a similar
model, Daniels et al. (2000) indicated that while L. monocytogenes attaches to and invades both enterocytes and
M-cells, no preferential targeting of M-cells occurs. However, they did not examine translocation of the monolayer.
While elegant work has unravelled the specific mechanisms by which L. monocytogenes directly invades host cells
(Hamon et al., 2006; Pentecost et al., 2006), there is much
evidence to support a role for M-cells in sampling the
pathogen from the intestinal lumen leading to rapid translocation to internal organs (Pron et al., 1998; Corr et al., 2006).
Viruses
Several viruses are transported by M-cells and reovirus
type-1, poliovirus and HIV type 1 (HIV-1) bind specifically
(Sicinski et al., 1990; Amerongen et al., 1991, 1994).
Reovirus, an orally transmitted murine pathogen, affects
the nervous system, causing encephalitis. Reovirus type-1
selectively adheres to ileal and colorectal M-cells, and this
is mediated by its outer capsid proteins (Amerongen et al.,
1994). Gastrointestinal transit causes proteolytic cleavage of
the outer capsid proteins, which become activated for
adherence (Helander et al., 2003). The receptors for reovirus
type-1 are a-2-3-linked sialic acid glycoconjugates that bind
the viral haemaglutinin sigma 1 (Davis & Owen, 1997).
Infection causes depletion of M-cells from the FAE, which
may affect host antiviral responses; however, the virus is
cleared within 10 days in mice by production of antiviral
sIgA (Silvey et al., 2001).
Poliovirus is the causative agent of poliomyelitis and
infects humans by the oral route. It is thought that the
primary sites of replication in the gut are PP (Sicinski et al.,
1990). In human tissues infected with poliovirus, virions
were found to be specifically adhering to the surface of
M-cells and in vesicles within M-cells (Sicinski et al., 1990).
Poliovirus type-1 and the corresponding attenuated vaccine
strain, Sabin, both translocate across M-cells in vitro (Ouzilou et al., 2002).
Transmission of HIV-1 infection via anorectal, cervicovaginal, foreskin and urethral epithelia accounts for 80% of
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8
AIDS cases (Amerongen et al., 1991). HIV-1 must cross the
mucosal barrier of the intestinal or genital tracts to infect
CD41 T-cells and translocation across epithelial cells or
M-cells of FAE of intestinal and tonsil lymphoid follicles
may play a role in infection (Amerongen et al., 1991).
Infection is thought to occur when epithelial barriers are
damaged, although studies in monkeys with simian immunodeficiency virus (SIV) have shown that infection can
occur via intact rectal mucosa (Amerongen et al., 1991).
HIV-1 has been shown to adhere to M-cells but not
enterocytes on villi or in the FAE in rabbits and mice
(Amerongen et al., 1991). Recently, it was shown that HIV1 is transported across M-cells via lactosyl cerebroside and
the chemokine receptor, CXCR4 (Fotopoulos et al., 2002).
They demonstrated that a lymphotropic (X4, syncytiuminducing HIV-1) HIV-1 strain crosses M-cells via lactosyl
cerebroside and CXCR4, receptors that are expressed apically on M-cells. A monotrophic (R5, nonsyncytium inducing) HIV-1 strain was unable to cross M-cells because they
do not express its CCR5 receptor; however, transfection of
M-cells with CCR5 cDNA restored translocation.
Prions
Prion disorders affect the mammalian brain by causing
plaques and lesions. Prion diseases include scrapie in sheep,
bovine spongiform encephalopathies (BSE or mad cow
disease) and the human disease, Creutzfeldt–Jakob disease.
These are all caused by an infectious misfolded prion (PrPSc)
that converts normal cellular prions to an abnormal form
(Heppner et al., 2001). Transmission of BSE to humans via
dietary exposure has become a concern since the appearance
of a new variant of Creutzfeld–Jakob disease (Heppner et al.,
2001). Using an in vitro M-cell model, it was shown that
Rocky mountain laboratory scrapie prions are transported
by M-cells while no transcytosis was observed in enterocytes
(Ghosh, 2002). Follicular DCs have been shown to be
important for development of prion pathogenesis and
subsequent infection of the neural system and this supports
the role of M-cells in prion transport (Ghosh, 2002).
An in vitro M-cell model
The study of M-cells has been problematic due to the
difficulties associated with isolating M-cells. M-cells have
been difficult to identify and isolate due to their low
numbers in the FAE, lack of an enriched preparation and
phenotype variation between species. M-cells have been
difficult to characterize biochemically and so little is known
about their cell biology. In the past, studies have relied on
in vivo or in situ methods such as isolated tissue loops or
explant cultures. However, the development of an in vitro
M-cell/FAE model has provided a reproducible approach in
which phenotypic and physiological features of the FAE and
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S.C. Corr et al.
M-cells overlying PP are maintained (Kerneis et al., 1997;
DesRieux et al., 2007). This model facilitates the study of
antigen transport across M-cells, and their interaction with
lymphocytes, bacteria and vaccines (Kerneis et al., 1997).
This system uses the human adenocarcinomal enterocytelike cell line, Caco-2, to mimic M-cell activity through
differentiation of epithelial enterocytes to M-cells via coculture with PPL or a Raji human lymphocyte cell line. Briefly,
the M-cell model involves a two-chamber transwell system
in which epithelial enterocytes are grown on a polycarbonate
porous membrane until differentiation to a villous-expressing cell type. Upon differentiation, freshly isolated PPL are
introduced into the basolateral chamber, where they migrate
into the monolayer (Fig. 3). This coculture induces the
M-cell phenotype (Kerneis et al., 1997). The efficiency of the
system has been determined by its ability to mimic key
M-cell properties. Translocation of fluorescein isothiocyanate (FITC)-labelled microspheres and live noninvasive
V. cholerae from the apical to basolateral chamber of
cocultures, are significantly increased in Caco-2 cells grown
in the presence of PPL, indicative of the sampling process
(Kerneis et al., 1997). Particle transport is temperaturedependent in cocultures indicating that a transcytotic route
is involved (Tyrer et al., 2002). Apical expression of alkaline
phosphatase is downregulated in cocultures and a redistribution of the actin-associated protein villin from the apical
surface to the cytoplasm indicates a loss of an organized
brushborder. a5b1 Integrin, normally expressed on the
lateral and basolateral surfaces of Caco-2 cells, is present on
apical membranes of cocultures (Schulte et al., 2000). The
M-cell in vitro model has shown great potential in the study
of the interaction of pathogens with the intestinal epithelium and also the determination of molecular mechanisms
required for translocation across M-cells (Schulte et al.,
2000). It also allows a method for determination of the
factors and mechanisms that influence M-cell development
and the development of oral vaccine delivery.
The future of M-cell research: vaccine
delivery
Oral vaccines will have to overcome many hurdles in order
to be effective. The hurdles include (1) poor accessibility of
mucosal DCs due to dilution or degradation of the antigen,
peristalsis and the mucus barrier, and (2) oral tolerance,
which downregulates cell-mediated and humoral immune
responses (Nicoletti, 2000). Given the unique features of
M-cells and their specialized ability to transcytose numerous
microorganisms and particulates, targeted delivery of antigens to M-cells may provide a mechanism for the efficient
presentation of antigens to initiate a mucosal immune
response (Brayden & Baird, 2004; Brayden et al., 2005). The
adhesins required for attachment of pathogens to specific
FEMS Immunol Med Microbiol 52 (2008) 2–12
9
M-cells
Fig. 3. Outline of the in vitro M-cell model
(Corr et al., 2006).
receptors on mucosal surfaces are being used for vaccine
delivery via antigen-encapsulated microspheres (Wu et al.,
2001; Byrd et al., 2005; Suzuki et al., 2006). Rotavirus
selectively binds to rabbit M-cells via the viral haemagglutinin adhesin, a-1 protein. Administration of rotavirus
protein a-1 conjugated with polylysine produces antigenspecific serum IgG and mucosal IgA responses (Kim et al.,
2002). Biodegradable microparticles made from the copolymer poly-(DL-lactide-coglycide) (PLG) also show potential
as mucosal delivery vehicles. PLG microspheres containing
rotavirus antigen VP-6 DNA confer protection against
challenge for up to 12 weeks. Microspheres coated with VP6 are selectively taken up by sheep PP and induce VP-6specific IgA following oral immunization of mice (Kim
et al., 2002). PLG microparticles containing HIV peptides
in combination with Ulex europaeus-1 lectin, which binds
M-cell apical surfaces, generate both mucosal and systemic
immune responses following intranasal immunization of
mice (Manocha et al., 2005). A recent study has demonstrated efficient uptake of PEGylated PLGA-based nanoparticles displaying surface RGD molecules that successfully
targeted b1 integrins on human M cells in cell culture and
murine M cells in vivo (Garinot et al., 2007).
While particle uptake has been demonstrated in vivo in
rodents and rabbits, it is unclear whether this is the case
in humans. Human oral vaccine Phase 1 trials using E. coli
FEMS Immunol Med Microbiol 52 (2008) 2–12
enterotoxigenic E. coli (ETEC) antigens contained in biodegradable microspheres have been carried out but had poor
outcomes and did not sufficiently stimulate mucosal immunity (Katz et al., 2003). Several pathogens target M-cells
as a mode of entry into the host and are being genetically
engineered to deliver antigen to mucosal inductive sites for
induction of antigen-specific immune responses (Suzuki
et al., 2006). Live-attenuated strains that have successfully
been used as mucosal vaccines include the Sabin strain of
poliovirus and Salmonella typhi Ty21a for polio and typhoid, respectively (Clark et al., 2001a, b). Investigation into
the use of Salmonella species as live vectors for delivery of
antigens and DNA is underway and one mutant lacking
DNA adenine methylase has been shown previously to be
unable to induce M-cell cytotoxicity but has an enhanced
ability to promote an immune response (Clark et al.,
2001a, b). Targeting ligand-coated particles to M-cells, combined with attempts to mimic pathogen entry routes via Mcells, could lead to increased uptake in vivo and successful
initiation of mucosal immune responses.
Conclusion
Despite relatively few numbers of M-cells within the intestinal epithelium compared with normal intestinal enterocytes, the nature of M-cells provides them with an
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10
important role in antigen sampling, bacterial translocation
and initiation of mucosal immune responses. With the
development of the in vitro M-cell model, research into
these important immune cells has improved. However, the
identification of specific M-cell antibodies and markers will
significantly improve one’s understanding of these cells.
Furthermore, thorough research into the relationship between M-cells and the establishment of disease, and their
ability to deliver antigen directly to mucosal immune initiation sites will improve delivery of existing mucosal vaccines
and development of new strategies for oral vaccines.
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